This application focuses on a key problem in radiation biology, elucidating molecular determinants of cell cycle checkpoint arrest in response to DNA damage. Our model system is the G1 delay induced by ionizing radiation of the budding yeast S. cerevisiae. Irradiated yeast cells remain arrested without initiating DNA replication until repair is initiated. Checkpoints and their molecular mechanisms are broadly conserved and represent an important component of the response to ionizing radiation. Definition of the pathways mediating this yeast response promises broad insight into homologous mechanisms in human cells. Preliminary studies. Previous studies of lethal X-irradiation of budding yeast revealed the Rad9 G2/M checkpoint in which DNA damage induces cell cycle arrest. In concert with other checkpoint factors, DNA damage activates the ATM homolog Mec1 to phosphorylate the BRCT protein Rad9 and activate unknown downstream inhibitors of cell cycle progression. We recently isolated radiation-sensitive mutations in the cyclin-dependent kinase Cdc28 that preserve essential functions in S phase and mitosis but abrogate arrest after lethal irradiation. We suggest that Cdc28 is the key cell cycle target of the Rad9 pathway. During this work we found that lethal gamma irradiation (greater than 1500 Gy) can yield a previously undescribed terminal G1 arrest. Moderate gamma irradiation (50-500 Gy) down-regulates Cdc28 kinase activity to induce a dose-dependent G1 delay that requires elements of the Rad9 pathway. Some radiation-sensitive Cdc28 mutants are specifically defective in G1 arrest and enter S phase without regard to the extent of DNA damage. These data suggest a yeast pathway analogous to DNA damage dependent regulation of CDK2/cyclin E by ATM, p53 and p21 in mammals. Hypothesis. We hypothesize that the Cdc28 cyclin-dependent kinase is the key cell cycle target of the G1 checkpoint signal. We propose to identify the molecules linking the DNA damage checkpoint proteins to Cdc28.
Specific Aims. We will 1) Further characterize the G1 checkpoint by physiological, biochemical and gene expression assays; 2) Analyze regulation of Cdc28 by studies of the radiation-sensitive Cdc28 mutants; and 3) Isolate cell cycle arrest regulators by genetic suppression of G1 checkpoint-defective Cdc28 mutants. Significance. G1 responses to DNA damage are often lacking in cancer cells, such as p53 mutants. The powerful genetic and biochemical tools available in yeast will facilitate rapid identification of novel genes mediating the G1 checkpoint response. Human homologs of these yeast genes may perform related functions.
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